An important need in cancer chemotherapy is a highly predictive in vitro chemosensitivity assay. A major impediment to the development of such a test had been the lack of the proper system to culture human tumors in vitro in such a way that they represnt tumors in the in vivo state. To overcome this problem, we have recently developed a tumor culture system for the in vitro growth of diverse human tumors in an in vivo-like way, allowing over 75% of explanted tumors to be grown, for periods of time that average over 90 days (Freeman, A.E. and Hoffman, R.M. Proc. Natl. Acad. Sci. USA, in press). The tumors are explanted on gels derived from pigskin rich in extracellular-matrix basement-membrane components such as various collagens and laminin. We have demonstrated that this tumor culture system allows the ready measurement of DNA and protein synthesis in individual cells within tumors, by autoradiographic techniques. We propose here to utilize this methodology of human tumor culture to determine the critical parameters for in vitro-drug sensitivity testing with the eventual goal of determining which parameters are the most predictive of the in vivo response of tumors to drugs. In this light, chemotherapeutic drugs will be tested for their effect on DNA synthesis, protein synthesis and cell viability staining in a quantitative manner. In concert with the above experiments, we will use antibodies to determine specific cell-type markers within the various types of human tumor cultures growing in vitro in order to identify the essential nature of cells within the tumors which is especially critical in measuring drug sensitivities. Tumors of the breast, lung and colon will be emphasized. Patient response data will be obtained and correlated to in vitro chemosensitivities to determine the most predictive in vitro prameters. The experiments proposed here should allow a major advance toward te goal of a commercial drug sensitivity test to be offered by Anticancer, Inc.